Substituted 3-cephem compounds as antibacterial agents

ABSTRACT

Certain 7-beta-[2-(2-amino-4-thiazolyl)-(Z)-2-(chloromethylene acetamido]-3-[(substituted)methyl]-ceph-3-em-4-carboxylic acids, pharmaceutically-acceptable salts thereof and conventional in vivo hydrolyzable esters thereof are valuable as broad spectrum antibacterial agents, particularly useful for the treatment of bacterial infections in man and other mammals.

BACKGROUND OF THE INVENTION

The present invention is directed to certain7-beta-[2-(2-amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(substituted)methyl]ceph-3-em-4-carboxylicacids, pharmaceutically-acceptable salts thereof and conventional invivo hydrolyzable esters thereof. These compounds are valuable as broadspectrum antibacterial agents, particularly useful for the systemictreatment of bacterial infections in man and other mammals; and, in theacid or salt form, as topical or industrial antibacterial agents.

The present invention employs a substantial number of the same7-amino-3-(substituted)methyl]ceph-3-em-4-carboxylic acid precursors asthose employed by Nagakura et al., published Japanese application61-037788, in the synthesis of certain 7-beta-[D-2-((4-ethyl-2,3-dioxo-1-piperazino)carbonylamino)-alpha-2-(4-hydroxyphenyl)acetamido]-3-[(substituted)methyl]-3-cephem-4-carboxylicacids. Japanese published applications 58-164593 and 60-11490 describe7-[2-(2-amino-4-thiazolyl)-2-(chloromethylene)acetamido]-3-methylceph-3-em-4-carboxylicacid and a number of analogs substituted on 3-methyl with a variety ofdiaza- and polyaza-heteroarylthio groups.

SUMMARY OF THE INVENTION

The present invention is directed to antibacterial cephem compoundshaving the formula ##STR1## wherein R is hydrogen or a radical forming agroup which is hydrolyzable under physiological conditions;

Y is ##STR2## R¹ and R² are taken separately, and R¹ is hydrogen,halogen, hydroxy, (C₁ -C₄)-alkoxy or (C₁ -C₄)alkyl, and

R² is hydrogen or (C₁ -C₄)alkyl; or

R¹ and R² are taken together and attached to adjacent carbons, and forma methylenedioxo group;

R³ is hydrogen or hydroxy; with the proviso that at least one of R¹, R²and R³ is other than hydrogen, halogen or (C₁ -C₄)alkyl;

X is hydrogen, halogen, --CH₂ COOH, --CH₂ NH₂ or --CH₂ NHCOR⁴ ;

R⁴ is (C₁ -C₄)alkanoyl or (C₁ -C₄)alkanoyl substituted with halogen; and

n is 1 or 2;

a pharmaceutically-acceptable acid addition salt, or apharmaceutically-acceptable cationic salt when R is hydrogen.

The term halogen, as used herein, refers to fluoro, chloro and bromogroups. Alkyl groups are straight chain or branched.

Pharmaceutically-acceptable acid addition salts include, but are notlimited to, those with hydrochloric acid, sulfuric acid, nitric acid,phosphoric acid, citric acid, maleic acid, succinic acid,benzenesulfonic acid, p-toluenesulfonic acid, 2-naphthalenesulfonic acidand methanesulfonic acid. Pharmaceutically-acceptable cationic saltsinclude, but are not limited to, those of sodium, potassium, calcium,N,N'-dibenzylethylenediamine, N-methylglucamine (meglumine) anddiethanolamine. The preferred cationic salts are those of potassium andsodium.

The reference to esters which are hydrolyzable under physiologicalconditions refers to those esters frequently referred to as in vivohydrolyzable esters or "pro-drugs". Such esters are now as well-knownand common in the penicillin art as pharmaceutically-acceptable salts.Such esters are generally used to enhance oral absorption, but in anyevent are readily hydrolyzed in vivo to the parent acid. The morepreferred ester forming radicals are those wherein R is:

(5-methyl-1,3-dioxol-2-on-4-yl)methyl,

1H-isobenzofuran-3-on-1-yl,

gamma-butyrolacton-4-yl,

--CHR⁶ OCOR⁷ ; or

--CHR⁶ OCOOR⁷ ;

wherein R⁶ is hydrogen or methyl; R⁷ is (C₁ -C₆)alkyl. The mostpreferred radicals are pivaloyloxymethyl and 1-(ethoxycarbonyloxy)ethyl.

Because of their ease of preparation and excellent antibacterialactivity, preferred compounds have Y as 3-fluoro-4-hydroxyphenyl,3,4-dihydroxyphenyl, 3-carboxy-4-hydroxyphenyl, 5-chloro-2-thienyl,5-(aminomethyl)-2-thienyl, 1,1-dioxo-3-thiolanylthio orcis-1-oxo-3-thiolanylthio.

The present invention is also directed to corresponding pharmaceuticalcompositions and a method of treating bacterial infections in mammals,including man.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is readily carried out. The compounds of theformula (I) are best formed by coupling formylated side chain acid ofthe formula ##STR3## with a 7-aminocephem compound of the formula##STR4## where R and Y are as defined above, to form an intermediatecompound of the formula ##STR5## which, with or without isolation, isdeformylated under mild solvolysis conditions to form the desiredcompound of the formula (I). When R is an ester group, said group can bealready in place in the intermediate (IV) or alternatively introducedafter coupling (and usually deformylation) have been accomplished.

The coupling step is accomplished by any one of a number of conventionalmethods well known in the beta-lactam and polypeptide arts. In thepresent invention, the preferred method employs a so-called Vilsmeierreagent, formed in situ from dimethylformamide and phosphorusoxychloride: ##STR6## carried out in an anhydrous, aprotic,reaction-inert solvent, generally at a temperature below ambient, e.g.,at about -25° to +10° C. The expression reaction-inert solvent, as usedhere and elsewhere herein refers to a solvent which does not interactwith starting materials, reagents, intermediates or product in a mannerwhich adversely affects the yield of the desired product. Methylenechloride is particularly well suited for this and subsequent stages ofthe present coupling process. Without isolation, the Vilsmeier reagentis then employed to activate the side chain acid, in the same solvent,but now generally at lower temperature, e.g., at about -15° to -30° C.When R is other than an ester forming radical, or the group Y contains acarboxylic acid function, the 7-aminocephem compound (III) is firstprotected as a silyl ester, conveniently usingbis(trimethylsilyl)acetamide as reagent. This will generally be done inthe same reaction-inert solvent as that employed for activation of theside chain, usually at a temperature of about 5° to 35° C., ambienttemperature being most convenient. The resulting solution is chilled andcombined with the activated side chain, generally in the sametemperature range employed to activate side chain, to form theintermediate N-formyl compound (IV), retaining any silyl esterprotecting groups. The intermediate (IV) is optionally isolated, withfacile removal of any silyl ester groups during the aqueous and/oralcoholic work-up. More convenient is to simply warm the couplingreaction to room temperature, dilute with methanol and allowdeformylation to proceed in the resulting acid solution. If theintermediate (IV) is isolated, it is simply deformylated in acidmethanol as a separate step.

The pharmaceutically-acceptable cationic salts of the present inventionare readily prepared by standard methods. For example, an equivalent ofthe corresponding cationic hydroxide, carbonate or bicarbonate or of anamine, is combined with the carboxylic acid in an organic or aqueoussolvent, preferably at reduced temperature (e.g., 0°-5° C.), withvigorous agitation and slow addition of the base. The salt is isolatedby concentration and/or the addition of a non-solvent. The salt isalternatively isolated directly from a reaction mixture, i.e., withoutisolation of the free acid, otherwise using similar techniques.

Likewise pharmaceutically-acceptable acid addition salts of the presentinvention are readily prepared by standard methods. For example, anequivalent of the acid is combined with the free amine form of thecompound in an organic or aqueous organic solvent. The salt is isolatedby concentration and/or the addition of a non-solvent. The salt isalternatively isolated directly from a reaction mixture, i.e., withoutisolation of the free amine, otherwise using similar techniques.

The compounds of the formula (I) wherein R represents a radical formingan in vivo hydrolyzable ester are prepared from the corresponding freeacids or cationic salts according to known methods, readily identifiedby those skilled in the penicillin art (see for example U.S. Pat. Nos.3,951,954, 4,234,579; 4,287,181; 4,342,693; 4,452,796; 4,342,693;4,348,264; 4,416,891; and 4,457,924). Methods of preparation areexemplified below. If desired, an ester containing a basic amine orcarboxylic acid function is converted to an acid addition salt orcationic salt, respectively, according to the methods of the immediatelypreceding paragraphs.

The required starting materials of the formulas (II) and (III) areprepared from known and available materials according to the preparativemethods which are specifically exemplified below.

As indicated above, the compounds of the formula (I) are useful asantibacterial agents. Such activity is demonstrated in vitro bymeasuring the minimum inhibitory concentrations (MIC's) in mcg/mlagainst a variety of microorganisms for those compounds where R ishydrogen. The procedure which is followed is the one recommended by theInternational Collaborative Study on Antibiotic Sensitivity Testing(Ericcson and Sherris, Acta. Pathologica et Microbiologia Scandinav,Supp 217, Section B: 64-68 [1971]), and employs brain heat infusion(BHI) agar and an inocula replicating device. Overnight growth tubes arediluted 100 fold for use as the standard inoculum (20,000-10,000 cellsin approximately 0.002 ml. are placed on the agar surface; 20 ml. of BHIagar/dish). Twelve 2 fold dilutions of the test compound are employed,with initial concentration of the test drug being 200 mcg/ml. Singlecolonies are disregarded when reading plates after 18 hours at 37° C.The susceptibility (MIC) of the test organism is accepted as the lowestconcentration of compound capable of producing complete inhibition ofgrowth as judged by the naked eye. Such measurements on the compounds(I) wherein R is hydrogen are, of course, determinative of the inherentantibacterial activity of those compounds where R forms a conventionalin vivo hydrolyzable ester.

Those compounds of the formula (I) having said in vitro antibacterialactivity are thus useful as industrial antimicrobials, for example inwater treatment, slime control, paint preservation and woodpreservation, as well as for topical application in mammals. In the caseof use of these compounds for topical application, it is oftenconvenient to admix the active ingredient with a non-toxic carrier, suchas vegetable or mineral oil or an emollient cream. Similarly, it can bedissolved or dispersed in liquid diluents or solvents such as water,alkanols, glycols or mixtures thereof. In most instances it isappropriate to employ concentrations of the active ingredient of fromabout 0.1 percent to about 10 percent by weight, based on totalcomposition.

In determining such in vivo activity of the compounds of the formula(I), acute experimental infections are produced in mice by theintraperitoneal inoculation of the mice with a standardized culture ofthe test organism suspended in 5 percent hog gastric mucin. Infectionseverity is standardized so that the mice receive a lethal dose of theorganism (the lethal dose is the minimum inoculum of organism requiredto consistently kill 100 percent of the infected, non-treated controlmice). The test compound of the formula (I) is administered at variousdosage levels, p.o. or i.p., to groups of infected mice. At the end ofthe test, the activity of the mixture is assessed by counting the numberof survivors among treated animals at a given dose. Activity isexpressed as the percentage of animals which survive at a given dose, orcalculated as PD₅₀ (dose which protects 50% of the animals frominfection). Preferred for oral (p.o.) use are those compounds of theformula (I) wherein R represents an in vivo hydrolyzable ester; whilefor parenteral (i.p.) use, those compounds wherein R is hydrogen arepreferred.

When using one of the present antibacterial compounds of the formula (I)for systemic control of bacterial infections in a mammal, particularlyman, the compound is administered alone, or mixed with pharmaceuticallyacceptable carriers or diluents, in the form of unit dosage forms fororal or parenteral use. For oral use, in vivo hydrolyzable esters aregenerally preferred, while for parenteral use (e.g., i.m. or i.v.), acidor salt forms are generally preferred. The daily dosage will be similarto those of other clinically useful beta-lactam antibiotics. Althoughthe prescribing physician will ultimately decide the dosage to be usedin a human subject, these compounds will normally be used orally atdosages in the range from about 20 to about 100 mg. per kilogram of bodyweight per day, and parenterally at dosages from about 10 to about 100mg. per kilogram of body weight per day, usually in divided doses. Insome instances, the prescribing physician will determine that dosagesoutside these limits are needed.

For oral administration, tablets, capsules, lozenges, troches, powders,syrups, elixirs, aqueous solutions and suspension, and the like areused, in accordance with standard pharmaceutical practice. Theproportional ratio of active ingredient to carrier will naturally dependon the chemical nature, solubility and stability of the activeingredient, as well as the dosage contemplated. In the case of tabletsfor oral use, carriers which are commonly used include lactose, sodiumcitrate and salts of phosphoric acid. Various disintegrants such asstarch, and lubricating agents, such as magnesium stearate, sodiumlauryl sulfate and talc, are commonly used in tablets. For oraladministration in capsule form, useful diluents are lactose and highmolecular weight polyethylene glycols, e.g. polyethylene glycols havingmolecular weights of from 2000 to 4000. When aqueous suspensions arerequired for oral use, the active ingredient is combined withemulsifying or suspending agents. If desired, certain sweetening and/orflavoring agents can be added. For parenteral administration, whichincludes intramuscular, intraperitoneal, subcutaneous, and intravenousinjection, sterile solutions of the active ingredient are usuallyprepared, and the pH of the solutions are suitably adjusted andbuffered. For intravenous use, the total concentration of solutes shouldbe controlled to render the preparation isotonic

The present invention is illustrated by the following examples. However,it should be understood that the invention is not limited to thespecific details of these examples.

EXAMPLE 1 Step 1

7-beta-[2-(2-Formylamino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido-3-[(3-fluoro-4-hydroxyhydroxyphenyl)methyl]ceph-3-em-4-carboxylicAcid

Under nitrogen, 12.7 ml of methylene chloride and 2.2 ml of anhydrousdimethylacetamide were cooled with stirring to 0° C. POCl₃ (1.58 ml,2.64 g, 17.2 mmols) was added, the mixture stirred at 0° C. for 30minutes and then cooled to -20° to -23° C., and finally2-(2-formylamino-4-thiazolyl)-(Z)-2-(chloromethylene)acetic acid (2.09g, 9 mmols) was added in 4 portions. The reaction mixture was maintainedat -20° to -23° C. while in a separate flask also under nitrogen,7-beta-amino-3-[(3-fluoro-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylicacid (2.5 g, 7.7 mmols) was suspended in 25 ml of CH₂ Cl₂ at roomtemperature, and 6.65 ml of bis(trimethylsilyl)acetamide (BSA) added.The resulting clear solution was stirred for 1 hour at room temperatureand then added to the cold reaction mixture and reacted at -20° to -23°C. for 1 hour. The reaction was quenched by the addition of 60 ml ofmethanol and concentrated to an oily residue, which was distributedbetween 350 ml ethyl acetate and 175 ml H₂ O, with the pH adjusted to2.5 by the addition of saturated NaHCO₃. The organic layer wasseparated, washed with 100 ml of water, dried over anhydrous sodiumsulfate and evaporated to dryness to give title product as a solid 2.68g, used directly in Step 2.

Step 2

7-beta-[2(2-Amino-4-thiazolyl)-(Z)-2-(chloro-methylene)acetamido]-3-[(3-fluoro-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylicAcid

Crude formyl product prepared according to Step 1 (2.93 g) was dissolvedin 100 ml of methanol, 1.5 ml (18 mmols) of concentrated HCl added, andthe mixture stirred at room temperature for 3 hours, then held 18 hoursat 4° to 5° C., at which time H₂ O (120 ml) was added and the mixtureconcentrated to 1/2 volume. The pH of the resulting slurry was made 7 bythe addition of saturated NaHCO₃ and the resulting solution clarified byfiltration. The pH of the filtrate was adjusted to 3.5 by the additionof 6N HCl and 1N HCl. Solid was collected by a filtration, washed withwater and dried to give 1.44 g of crude product. Purification wasachieved by suspending the crude product (1.2 g) in 30 ml of water,adjusting the pH to 8.2 with saturated NaHCO₃, and extracting with 20 mlof ethyl acetate. The aqueous layer was separated, the pH readjusted to3.5 with 1N HCl and purified title product collected by filtration,washed with water and dried in vacuo overnight. Yield: 760 mg; m.p.189°-190° C.(dec.); ir (KBr) 3316, 1764, 1629, 1514, 1439, 1364, 1290,1237, 1183, 1112, 1060, 1011, 952, 900, 816, 791, 728, 582, 204 cm⁻¹.Nmr (DMSO-d₆) 5.05 (d, C₆ -H), 5.63 (q, C₇ -H).

EXAMPLE 27-beta-[2-(2-Amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(5-chloro-2-thienyl)-methyl]ceph-3-em-4-carboxylicAcid

Under nitrogen, 1.27 ml of CH₂ Cl₂ and 0.22 ml of anhydrousdimethylacetamide was cooled to 0° C. POCl₃ (0.213 ml) was added, andthe mixture stirred at 0° C. for 30 minutes, then cooled to -20° to -23°C. 2-(2-Formylamino-4-thiazolyl)-(Z)-2-(chloromethylene)acetic acid (272mg) was added and the mixture stirred for 10 minutes at -20° to -23° C.Meanwhile, in a separate flask,7-beta-amino-3-[(5-chloro-2-thienyl)methyl]ceph-3-em-4-carboxylic acid(330.8 mg) was suspended in 2.5 ml of methylene chloride at roomtemperature, 0.863 ml of bis(trimethylsilyl)acetamide (BSA) was added,and the resulting solution stirred at room temperature for 1 hour, addedto the cold reaction mixture at -20° to -23° C. and reacted at thattemperature for 1 hour. The mixture was warmed up to room temperature, 6ml of methanol was added, and the mixture was stirred at roomtemperature for 2 hours to complete deformylation. Finally, the reactionmixture was concentrated to an oily residue which was dissolved in 10 mlof ethyl acetate and 5 ml of water. The pH of the mixture was adjustedto 7.5 with saturated NaHCO₃. The aqueous layer was separated, filtered,and the pH of the filtrate was adjusted to 3.0 by an addition of 6N and1N HCl. The mixture was filtered, the solids washed well with water anddried overnight in vacuo to yield title product, 140 mg; m.p. 184°-185°(dec.).

EXAMPLE 3 Step 17-beta-[2-(2-Formylamino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(1,1-dioxo-3-thiolanylthio)methyl]ceph-3-em-4-carboxylicAcid

By the method of Step 1 of Example 1,7-beta-amino-3-[(1,1-dioxo-3-thiolanylthio)methyl]ceph-3-em-4-carboxylicacid (312 mg, 0.85 mmol) was converted to crude, partially deformylatedtitle product, 179 mg.

Step 27-beta-[2-(2-Amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(1,1-dioxo-3-thiolanylthio)methyl]ceph-3-em.-4-carboxylicAcid

By the method of Step 2 of Example 1, present Step 1 product (170 mg)was converted to present title product, 104 mg; ir (KBr) 1774, 1665,1630 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) includes 9.6(m), 7.16 (broad s), 6.83 (s),6.40 (s), 5.74 (dd), 5.2 (d), 3.8-2.8 (unresolved multiplets), 1.96 (m).

EXAMPLE 47-beta-[2-(2-Amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(cis-1-oxo-3-thiolanylthio)methyl]ceph-3-em-4-carboxylicAcid

By the method of Example 2,7-beta-amino-3-[(cis-1-oxo-3-thiolanylthio)methyl]ceph-3-em-4-carboxylicacid (321 mg, 0.92 mmol) was converted to present title product, 79 mg;ir (KBr) 1772, 1659, 1634 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 9.6 (m, 1H) 7.15(broad s, 2H), 6.83 (s, 1H), 6.34 (s, 1H), 5.73 (dd, 1H), 5.21 (d, 1H),3.8-1.7 (unresolved multiplets).

EXAMPLE 57-beta-[2-(2-Amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(3-carboxy-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylicAcid

By the method of Example 2,7-beta-amino-3-[(3-carboxy-4-hydroxyphenyl)methyl]-3-cephem-4-carboxylicacid (0.35 g, 1 mmol) was converted to title product 315 mg, m.p.235°-237° C. (dec.); ir (KBr) 3367, 1774, 1661, 1544, 1489, 1442, 1366,1244 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.1-4.2 (m), 4.9 (d), 5.05 (d), 5.2 (d),5.8 (q), 6.4 (s), 6.8 (q), 7.1-7.4 (m), 7.6 (q), 9.6 (d).

EXAMPLE 67-beta-[2-(2-Amino-4-thiazolyl-(Z)-2-(chloromethylene)acetamido]-3-[(3,4-dihydroxyphenyl)methyl]ceph-3-em-4-carboxylicAcid

By the method of Example 2,7-beta-amino-3-[(3,4-dihydroxyphenyl)methyl]ceph-3-em-4-carboxylic acid(306 mg, 1 mmol) was converted to present title product, 407 mg; ¹ H-nmr(DMSO-d₆) 3.4-3.6 (m), 5.2 (d), 5.6-5.8 (dd), 6.4-6.8 (q), 7.1 (s), 8.75(d), 9.6 (d).

EXAMPLE 77-beta-[2-(2-Amino-4-thiazolyl)-(Z)-2-methylene)acetamido]-3-[(5-(2-chloroacetamidomethyl)-2-thienyl)methyl]-ceph-3-em-4-carboxylicAcid

By the method of Example 2,7-beta-amino-3-[(5-(chloroacetylaminomethyl)thienylmethyl]-ceph-3-em-4-carboxylicacid (0.802 g) was converted to present title product, 658 mg; m.p.152°-155° C.; ir (KBr) 3293, 3066,, 2954, 1765, 1657, 1366, 1232, 1182,1058, 1038 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.2-3.6 (m, CH₂), 4.07 (s, 2H), 4.37(d, CH₂), 5.17 (d, C₆ -H), 5.7 (q, C₇ -H), 6.8 (d), 7.1 (s), 8.8 (q),9.5 (d).

EXAMPLE 87-beta-[2-(2-Amino-4-thiazolyl]-(Z)-2-(chloromethylene)acetamido]-3-[5-(aminomethyl)-2-thienyl)methyl]ceph-3-em-4-carboxylicAcid

Title product of the preceding Example (640 gm) was suspended in amixture of 6.5 ml of CH₃ CN and 3.2 ml of water. Thiourea (246 mg) and220 mg of sodium acetate trihydrate were added, and the mixture wasstirred at room temperature for 18 hours, at which time 50 ml ofisopropyl alcohol was added dropwise. The resulting solids werefiltered, washed well with isopropyl alcohol and dried in vacuoovernight to yield 400 mg of the title product, m.p. 185°-187° (dec.),solvated with one molar equivalent of isopropyl alcohol (by nmr). Thelatter was suspended in a mixture of 20 ml of ethyl acetate and 10 ml ofwater and the pH was made 2 by the addition of 1N HCl. The aqueous layerwas separated and the pH adjusted to 3.1 with saturated NaHCO₃. Theresulting solid was collected by filtration, washed well with water anddried in vacuo overnight to yield title product, 90 mg; m.p. 197° -198°C. (dec.); ir (KBr) 3300, 3062, 1762, 1651, 1528, 1389, 1365, 1281,1231, 1184, 1056, 1016 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.2-3.6 (m), 3.9 (s),4.4 (d), 5.06 (d, C₆ -H), 5.66 (s, C₇ -H), 6.39 (s), 6.7 (q), 7.18 (s),9.0 (q), 9.5 (d).

EXAMPLE 9 Pivaloyloxymethyl7-beta-[2-(2-amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(3-fluoro-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylate

Title product of Example 1 (300 mg) in 1.5 ml anhydrousdimethylacetamide was combined with triethylamine (0.164 ml) andchloromethyl pivalate (0.17 ml) and reacted for 18 hours at roomtemperature. The reaction mixture was diluted with 2.5 ml H₂ O,extracted 2×4 ml ethyl acetate, and the extracts combined, backwashedwith 2.5 ml H₂ O, dried over MgSO₄ and evaporated to dryness. Theresulting residue (300 mg) was triturated and stirred with 4 ml ether,filtered and dried in vacuo to yield title product, 125 mg; ir (KBr)1780, 1755, 1665 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 1.16 (s, 9H), 2.05 (s, 3H),3.2-4.0 (m), 5.6-6.2 (m), 6.86 (s, 1H), 7.10 (dd), 9.55 (d).

By the substitution of an equivalent amount of alpha-chloroethyl ethylcarbonate for chloromethylpivalate, this method is used to produce1-(ethoxycarbonyloxy)ethyl7-beta-[2-(2-amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(3-fluoro-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylate.

By the substitution of an equivalent amount of the title product ofExample 4 for the title product of Example 1, this method is used toproduce pivaloyloxymethyl7-beta-[2-(2-amino-4-thiazolyl)-(Z)-2-(chloromethylene)acetamido]-3-[(cis-1-oxo-3-thiolanylthio)methyl]ceph-3-em-4-carboxylate.In like manner, the corresponding 1-(ethoxycarbonyloxy)ethyl ester isprepared by further substituting alpha-chloroethyl ethyl carbonate forchloromethylpivalate.

PREPARATION 1 Ethyl 2-Formyl-2-(2-formylamino-4-thiazolyl)acetate

Under nitrogen, 14 g (0.29 mmol) of 50% NaH in oil was washed with 2×40ml of hexane and then suspended in 90 ml of anhydrous tetrahydrofuran(THF). To this slurry at 0° C. was added dropwise a solution of ethyl2-(2-amino-4-thiazolyl)acetate (27 g, 0.145 mmol) in 290 ml of anhydrousTHF, followed by ethyl formate (21.5 g, 0.29 mmol), and the mixturestirred at room temperature for 18 hours. The pH of reaction mixture wasadjusted to 7 by the addition of 50% aqueous acetic acid and the mixtureextracted with 250 ml ethyl acetate. The aqueous layer was furtherextracted with 2×50 ml of 1:1 THF:ethyl acetate. The extracts werecombined, dried over Na₂ SO₄ and evaporated to a solid, which wasrecrystallized from ethyl acetate to yield purified title product, 17.8g (50.7%]; m.p. 179°-180° C.; ¹ H-nmr (CDCl.sub. 3 -d₆ -DMSO) 1.33 (t,3H), 4.16 (q, 2H), 7.50 (s, 1H), 8.00 (s, 1H), 8.53 (s, 1H).

PREPARATION 2

Ethyl2-(Chloromethylene)-2-[2-(dimethylaminomethyleneamino)-4-thiazolyl]acetate

Title product of the preceding Preparation (20 g, 82 mmols) wasdissolved in 100 ml of dimethylformamide. With tap water cooling, 15.6 g(100 mmols) of POCl₃) was added in 4 portions. The stirred mixture wasreacted at 100°±2° C. for 1.5 hours, cooled to room temperature, the pHadjusted to 7 with 6N NaOH and 1N NaOH and extracted with 2×150 ml ethylacetate. The combined organic extracts were dried over Na₂ SO₄,evaporated to yield title product as an oil (26.23 g), which consistedof a mixture of syn- and anti-isomers.

PREPARATION 3 Ethyl(Z)-2-(chloromethylene)-2-(2-formylamino-4-thiazolyl)acetate

To the title product of the preceding Preparation (26 g) was added 52.3ml of acetic formic anhydride reagent (Fieser and Fieser Reagents forOrg. Syn. Vol. 1, page 4) and the mixture stirred at room temperaturefor 4 hours. Water (200 ml) was added and the mixture concentrated invacuo to an oil. The oil was taken up in 200 ml ethyl acetate, washed1×100 ml saturated NaHCO₃ and 1×100 ml water, dried over Na₂ SO₄ andevaporated to dryness to give 20.24 g of solid, a mixture of syn- andanti-isomers. To separate the desired (Z)-isomer, the mixture (20 g) wasdissolved by warming in the minimum necessary CHCl₃ and chromatographedon 1 kg of silica gel with 5:1 CHCl₃ :ethyl acetate as eluant to yieldpurified title product, 12.17 g, m.p. 97°-99° C.

PREPARATION 4(Z)-2-(Chloromethylene)-2-(2-formylamino-4-thiazolyl)acetic Acid

The title product of the preceding Example (5.21 g, 20 mmols) wassuspended in 11 ml of tetrahydrofuran and cooled to 0° C., 3.37 g (60mmols) of KOH in 55 ml of water was added dropwise at 0° C., and theresulting mixture stirred at room temperature for 7 hours, thenextracted with 2×55 ml of 1:1 ethyl acetate:diethyl ether. The residualaqueous layer was cooled to 0° C., and the pH adjusted to 2.0 with 6NHCl and 1N HCl. The solid was collected by a filtration, washed withwater and dried overnight to give crude title product 3.93 g (85%), m.p.144°-145° (dec.) contaminated with free amine. This crude wasre-formylation by a treatment with 21 ml of acetic-formic anhydride atroom temperature for 4 hours, concentrated under in vacuo, and the solidresidue slurried in hexane, filtered and dried overnight in vacuo toyield 4 g of purified title product, m.p. 151°-152° C. (dec.).

PREPARATION 57-beta-Amino-3-[(3-fluoro-4-hydroxyphenyl)-methyl]ceph-3-em-4-carboxylicAcid

7-Aminocephalosporanic acid (7-ACA,7-beta-amino-3-(acetoxymethyl)ceph-3-em-4-carboxylic acid; 5.44 g, 20mmols) was dissolved in 40 ml of trifluoroacetic acid at 10° to 15° C.To the stirred solution was added borontrifluoride etherate (11.35 g, 80mmols) followed by 3.36 g (30 mmols) of 2-fluorophenol, maintaining 10°to 15° C. throughout both additions. The resulting mixture was stirredat 23° to 25° C. for 4.5 hours, then concentrated in vacuo to an oilysolid which was dissolved in 30 ml of water. The resulting solution wascooled in ice-bath and the pH adjusted to 3.5 by a dropwise addition of28% NH₄ OH. The resulting solids were collected by a filtration, washedwell with water, dried in vacuo overnight to yield title product, 6.35 g(98%), m.p. 170°-171° C.; ir (KBr) 1798, 1674, 1616, 1514, 1474 cm⁻¹ ; ¹H-nmr (DMSO-d₆) 9.63 (s, 1H), 6.6-7.2 (m, 3H), 4.87 (d, 1H), 4.66 (d,1H), 3.02-3.68 (m, 6H).

By the same method, substituting a molar equivalent of the appropriatephenol, aryl ether or thiophene for 2-fluorophenol, the followingadditional compounds were prepared:

7-beta-Amino-3-[(3,5-di(t-butyl)-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylicacid; 33% yield; ir (KBr) 1770 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 1.37 (s,C(CH₃)3), 3.0-4.5 (m, 3H), 4.72 (d, J═4,6Hz, 1H), 4.99 (d, J=4.6Hz, 1H),7.06 (2H).

7-beta-Amino-3-[(3,4-dihydroxyphenyl)methyl]-2-ceph-3-em-4-carboxylicacid; 61% yield; ir (KBr) 1770 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 2.80-4.20 (m,3H), 4.73 (d, J═5.0Hz, 1H), 5.00 (d, J═5.0Hz), 6.4-7.00 (m, 3H).

7-beta-Amino-3-[(3,5-dimethyl-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylicacid; 89% yield; ir (KBr) 1800 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 2.15 (s, arylCH₃, 6H), 3.12/3.46 (dd, J═17Hz, C₃ -CH₂), 3.46/3.76 (dd, J═14.6Hz), C₂-H₂, 4.74/4.76 (d, J═4.5, C₇ -H and C₈ -H), 6.83 (aryl H).

7-beta-Amino-3-[(2,4-dihydroxyphenyl)methyl]ceph-3-em-4-carboxylic acid;33% yield; ir (KBr) 1770 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 2.80-4.40 (m-C₂ H₂, C₃-CH₂), 4.75/4.95 (2d, J═5.0Hz, C₇ -H and C₈ -H), 6.05-7.05 (m,aromatic-H).

7-beta-Amino-3-[(2,5-dihydroxyphenyl)methyl]ceph-3-em-4-carboxylic acid;53% yield; ir (KBr) 1770 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.00-4.00 (m, C₂ -H₂,C₃ -CH₂), 4.75/5.02 (2d, J═5.0Hz, C₇ -H and C₈ -H), 6.40-7.00 (m,aromatic H).

7-beta-Amino-3-[(3-carboxy-4-hydroxyphenyl)methyl]ceph-3-em-4-carboxylicacid; 57% yield; ir (KBr) 3509, 1801 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 7.7 (q,1H), 7.41 (q, 1H), 6.8 (m, 3H), 3.3-5.1 (m, 6H), 2.0 (s, 1H).

7-beta-Amino-3-[(4-hydroxy-2-naphthyl)methyl]ceph-3-em-4-carboxylicacid; 81% yield; ir (KBr) 1780 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.10-3.60 (m, C₃-CH₂), 4.00-4.30 (s, C₂ -H), 6.70-8.40 (m, aromatic H).

7-beta-Amino-3-[(4-methoxyphenyl)methyl]ceph-3-em-4-carboxylic acid; 69%yield; ir (KBr) 1800, 1245 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.10-3.50 (dd,J═17.2Hz), C₃ -CH₂), 3.75 (s, OCH₃), 3.50-4.10 (m, C₂ -H), 4.73/5.00(2d, J═5.0Hz, C₇ -H and C₈ -H), 6.89-7.22 (dd, J═9.0Hz, aromatic H).

7-beta-Amino-3-[(3,4-methylenedioxyphenyl)methyl]ceph-3-em-4-carboxylicacid; 74% yield; ir (KBr) 1795, 1225 cm⁻¹ ; ¹ H-nmr (DMSO-d₆); 3.12.3.52(dd, J═17Hz, C₃ -CH₂), 3.45-3.87 (dd, J═16Hz, C₂ H), 4.75/5.03 (2d,J═5.0Hz, C₇ -H), 6.01 (s, --OCH₂ O), 6.60-7.10 (m, aromatic H).

7-beta-Amino-3-[3,4-dihydroxy-5-methoxyphenyl)methyl]ceph-3-em-4-carboxylicacid; 63% yield; ir (KBr) 1780 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.00-4.10 (m, C₂H, C₃ -CH₂), 3.70 (s, OCH₃), 4.72/4.96 (2d, J═5.0Hz, C₇ -H and C₈ -H),6.30-6.70 (m, aromatic H).

7-beta-Amino-3-[(4-hydroxy-3,5-dimethoxyphenyl)methyl)ceph-3-em-4-carboxylicacid; 99% yield; ir (KBr) 1795 cm⁻¹ ; ¹ H-nmr (DMSO-d₆), 3.0-4.10 (m, C₂-H, C₃ -CH₂), 3.72 (s, OCH₃), 3.80 (s, OCH₃), 4.75/4.97 (2d, J═5.0Hz, C₇-H and C₈ -H), 6.68 (s, aromatic H).

7-beta-Amino-3-(2-thienylmethyl)ceph-3-em-4-carboxylic acid; 85% yield;ir (KBr) 1790 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.20, 3.61 (dd, J═17Hz, C₃ -CH₂),3.50-4.20 (m, C₂ -H), 4.75/5.01 (2d, J═5Hz, C₇ -H and C₈ -H), 6.60-7.60(m, heteroaromatic hydrogen).

7-beta-Amino-3-[(5-bromo-2-thienyl)methyl]ceph-3-em-4-carboxylic acid;39% yield; ir (KBr) 1800 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.21, 3.65 (dd,J═17Hz, C₃ CH₂), 3.68, 4.05 (dd, J═15Hz, C₂ -H), 4.75 (d, J═5Hz), 5.00(d, J═5.0Hz), 6.85-7.08 (dd, J═3.6Hz, heteroaromatic H).

7-beta-Amino-3-[(5-(carboxymethyl)-2-thienyl)methyl]ceph-3-em-4-carboxylicacid; 77% yield; ir (KBr) 1800, 1710 cm⁻¹ ; ¹ H-nmr (DMSO-d₆) 3.23-3.62(dd, J═17Hz, C₃ -CH₂), 3.68-4: 05 (dd, J═16Hz, C₂ -H), 3.75 (s, --CH₂COOH), 4.73 (d, J═5Hz), 5.00 (d, J═5Hz), 6.80 (s, heteroaromatic H).

7-beta-Amino-3-[(5-(2-chloroacetylamino)methyl-2-thienyl)methyl]ceph-3-em-4-carboxylicacid; 99% yield; ir (KBr) 1800, 1650 cm⁻¹ ; 1H-nmr (DMSO-d₆) 3.25-3.60(dd, J═17Hz, C₃ -CH₂), 3.68-4.02 (dd, J═15Hz, C₂ -H), 4.10 (s, COCH₂Cl), 4.40 (d, J═5.5Hz, --CH₂ NH--), 4.75 (d, J═5Hz), 5.00 (d, J═5Hz),6.81 (s, heteroaromatic H), 8.78 (broad, --NHCO--).

7-beta-Amino-3-[(5-chloro-2-thienyl)methyl]ceph-3-em-4-carboxylic acid;96% yield; m.p. 188°-190° C. (dec.); ir (KBr) 1800, 1620, 795 cm⁻¹ ; ¹H-nmr (DMSO-d₆) 6.9 (d, 1H), 6.8 (d, 1H₀, 4.9 (d, 1H0, 4.7 (d, 1H), 3.9(d, 1H), 3.5-3.6 (q, 4H), 3.2 (d, 1H).

7-beta-Amino-3-[(4- and 2-hydroxyphenyl)methyl]ceph-3-em-4-carboxylicacid; 87% yield (o, p mixture); ir (KBr) 1780 cm⁻¹ ; ¹ H-nmr (DMSO-d₆)3.06-3.51 (dd, J═18Hz, C₃ -CH₂), 3.41-3.80 (dd, J═14Hz, C₂ H), 4.72-4.99(d, J═5Hz, C₇ -H, C₈ -H), 6.70-7.08 (q, J═8Hz, aromatic H).

PREPARATION 6 7-beta-Ais-(1-oxo-3-thiolanylthio)methyl]ceph-3-em-4-carboxylic acid

Under nitrogen, anhydrous acetonitrile (1.4 ml) was cooled to 5° C., 8ml of BF₃ OEt₂ was added and the mixture stirred at 5°-10° C. for 30minutes. Maintaining the same temperature range,cis-3-mercaptothiolane-1-oxide (806 mg) in 2.8 ml acetonitrile wasadded, followed by 7-ACA (942 mg) in portions. The temperature wasincreased and the mixture stirred 5 hours at room temperature, thenpoured into 40 ml of water and ice, the pH adjusted to 3.5, and titleproduct recovered by filtration, washed well with water, and dried toyield 1.1 g (91%) of title product; ir (KBr) 1800, 1620 cm⁻¹ ; ¹ H-nmr(DMSO-d₆) 5.02 (d, 1H), 4.76 (d, 1H), 3.45-3.81 (m), 2.2-2.9 (m).

By the same method, 3-mercaptothiolane-1,1-dioxide was converted to7-beta-amino-3-[(1,1-dioxo-3-thiolanylthio)methyl]ceph-3-em-4-carboxylicacid in 90% yield; m.p. 206°-208° C. (dec.); ir (KBr) 1803, 1619, 1170cm⁻¹ ; 1H-nmr (DMSO-d₆) 4.99 (2d, J═4.8Hz, 1H, reflecting racemic side),4.73 (d, J═4.8Hz, 1H), 3.8-2.8 (unresolved multiplet), 2.41 (m, 1H),1.94 (m, 1H).

What is claimed is:
 1. A cephem having the formula ##STR7## wherein R ishydrogen or a radical forming a group which is hydrolyzable underphysiological conditions;Y is ##STR8## R¹ and R² are taken separately,and R¹ is hydrogen, halogen, hydroxy, (C₁ -C₄)alkoxy or (C₁ -C₄) alkyl,andR² is hydrogen or (C₁ -C₄)alkyl; or R¹ and R² are taken together andattached to adjacent carbons, and form a methylenedioxo group; R³ ishydrogen or hydroxy; with the proviso that at least one of R¹, R² and R³is other than hydrogen, halogen or (C₁ -C₄)alkyl; and R⁴ is (C₁-C₄)alkanoyl or (C₁ -C₄)alkanoyl substituted with halogen;apharmaceutically-acceptable acid addition salt, or apharmaceutically-acceptable cationic salt when R is hydrogen.
 2. Acephem of claim 1 wherein R is hydrogen.
 3. A cephem of claim 2 whereinY is ##STR9##
 4. The cephem of claim 3 wherein Y is ##STR10##
 5. Thecephem of claim 3 wherein Y is ##STR11##
 6. The cephem of claim 3wherein Y is ##STR12##
 7. A cephem of claim 1 wherein Ris:(5-methyl-1,3-dioxol-2-on-4-yl)methyl, 1H-isobenzofuran-3-on-1-yl,gamma-butyrolacton-4-yl, --CHR⁶ OCOR⁷, or --CHR⁶ OCOOR⁷, wherein R⁶ ishydrogen or methyl and R⁷ is (C₁ -C₆)alkyl.
 8. A cephem of claim 7wherein R is 1-ethoxycarbonyloxyethyl.
 9. A cephem of claim 7 wherein Ris pivaloyloxymethyl.
 10. The cephem of claim 9 wherein Y is ##STR13##11. A pharmaceutical composition comprising an antibacterially effectiveamount of a compound of claim 1 and a pharmaceutically-acceptablecarrier.
 12. A pharmaceutical composition comprising an antibacteriallyeffective amount of a compound of claim 2 and apharmaceutically-acceptable carrier.
 13. A pharmaceutically compositioncomprising an antibacterially effective amount of a compound of claim 7.14. A method of treating a bacterial infection in a mammal whichcomprises treating said mammal with an antibacterially effective amountof a compound of claim
 1. 15. A method of treating a bacterial infectionin a mammal which comprises treating said mammal with an antibacteriallyeffective amount of a compound of claim
 2. 16. A method of treating abacterial infection in a mammal which comprises treating said mammalwith an antibacterially effective amount of a compound of claim 7.